Introductiontochemicalanalysis offorensicsamples
1Introduction
Forensicscienceisafieldinwhichscienceisappliedduringacriminalinvestigationfortheanalysisofevidenceobtainedthroughthecourseofinvestigation.Becauseitismainlyascientificfield,severalanalytical techniquesareusedfortheanalysisofvariousevidencefoundatthescene ofthecrime.Thepersonnelinvolvedinthecrimesceneinvestigation includethosewhoarepresentatthecrimescenesuchasdoctors,police, andothertechniciansresponsibleforthecollectionofevidence.Forensic scientistsworkinginthelaboratoryaremoreexperiencedwiththedifferent analyticaltechniquesusedfortheiranalysis.Biological,physical,andchemicalevidencearemostwidelyencounteredduringtheinvestigation.Priorto analysiswiththeanalyticaltechniques,thesesamplesareusuallypreppedin ordertoensureoptimalanalysis.Inthischapter,abriefoverviewofthedifferentanalyticaltechniquesusedfortheanalysisoftheevidenceencountered duringtheinvestigationisgiven.Thischapterwillprovideabasicoutlineof thevariousanalyticaltechniquesusedduringevidenceanalysis.
2Chemicalanalyticaltechniquesusedfortheanalysis ofevidence
Inthissection,spectroscopic,chromatographic,microscopic,andother emerginganalyticaltechniquesarediscussedinbrief. Fig.1 givesabrief overviewofthevariousforensicevidenceandtheanalyticaltechniquesused fortheiranalysis.
2.1Spectroscopictechniques
Spectroscopictechniquesarebasedontheinteractionofthesamplewith electromagneticradiation.Electromagneticradiationisaformofenergythat consistsoftheelectricalandthemagneticcomponentpropagatingatright anglestoeachother.Theelectromagneticradiationbasicallyhasadual
HandbookofAnalyticalTechniquesforForensicSamples © 2021ElsevierInc. https://doi.org/10.1016/B978-0-12-822300-0.00001-X Allrightsreserved.
Spectroscopic techniques
Forensic evidence
Illicit drugs Explosives
Chromatographic techniques
Biological fluids Paints and pigments
Microscopic techniques
Emerging analytical techniques Hair
Fig.1 Analyticaltechniquesfortheexaminationofforensicevidence.
nature,thatis,itcantravelineitherawaveformorinaparticulateform.The electromagneticradiationassuchconsistsofpacketsofenergyknownas photons.Itisduetotheseparticlesthattheelectromagneticradiationconsistsofboththewave-andparticle-likebehavior.Spectroscopicanalytical techniquesinvolvetheinteractionofmatterwiththeelectromagneticradiationofawavelengthrangefrom10 3 to10 8 nm,whichbasicallycovers theinfraredtoUV-visiblerange.InthissectionUV-visibleandfluorescence,infrared,nuclearmagneticresonance,Raman,andatomicabsorption andemissionspectrometry.
2.1.1UV-visibleandfluorescencespectroscopy
UV-visiblespectroscopyisatechniquethatmeasuresabsorptionoccurring intheUV-visiblerange.TheUVregionextendsfrom190to350nmwhile thevisiblerangeextendsfrom350to800nm.Themainprinciplebehind theUV-visibletechniqueistheelectronictransitionsthatoccurfromthe bondingtonon-andantibondingorbitals,thatis,fromn, π,andsigmato π* andsigma*,respectively.Suchtransitionsoccurincompoundsthatconsistofdoublebondsorarearomatic.Theabsorbancebysuchcompoundsin thisrangeisdeterminedbytheBeer-Lambertlaw,whichtakesintoaccount theconcentrationofthesamplewiththeintensityofthetransmittedlight. Throughthistechnique,itispossibletoanalyzemyriadcompounds.Fluorescencespectroscopyisatechniquethatalsotakesintoaccounttheelectronictransitionsfromthegroundstatetoahigherstate.Whenthe
sampleisirradiatedwithahighenergysource,itabsorbstheenergyandtransitionstoahigherstate.However,itlosesitsenergyandfallsbacktoits groundstatewhileemittingfluorescenceenergyforanoticeablyshort periodoftime.Thefluorescenceemittedischaracteristictoeachelement andisthereforecrucialfortheanalysisofvariousevidence.Thesespectroscopictechniquesareamongthemostubiquitoustoolsusedfortheanalysis ofvariousforensicevidencesuchasblood,semen,saliva,urine,andother bodyfluids;counterfeitfoodproducts;drugs;paints;orpetroleumproducts. Someofthesamplescanbedirectlyanalyzedthroughthistechniquewhile somerequireappropriatesamplepreparationtechniques(Altemose,1986; R€atyetal.,2004).
2.1.2Infraredandnear-infraredtechniques
Infraredandnear-Infraredtechniquesarevibrationalspectroscopictechniquesthattakeintoaccountthevibrationalfrequenciesofthemolecules whentheyareirradiatedbystronginfraredradiation.Theinfraredradiation rangesfrom700nmto1mm.Whentheradiationhitsthesample,themoleculeabsorbstheenergy,duetowhichthebondsstarttovibrate.The behaviorofthemoleculesatsuchastageisequivalenttothatofaspring andvibrationssuchasrocking,twisting,stretching,andbendingtakeplace. Thisvibrationalfrequencyischaracteristicofaparticularcompoundandcan thusbeeasilyusedforthedeterminationofthecompound.InfraredspectroscopyisusuallyusedinconjunctionwiththeFouriertransformtechnique,whichusesaMichelsoninterferometerandisbasedonthe constructiveanddestructiveinterferenceoftheradiationobtainedfrom thesample.ThefinalspectracanbeeasilydevelopedthroughFouriertransformation.Thistechniqueisnondestructiveandisincrediblyusefulforthe analysisofevidencesuchashair,blood,andotherbiologicalevidenceaswell asillicitandcounterfeitedproducts,explosives,andgunshotresidue.Nearinfraredspectroscopyisatechniquesimilartoinfraredspectroscopy,except thatitusesnear-IRradiationfortheanalysisofcompounds.TheNIRrange isfrom0.7to1.4 μm.Thistechniqueiswidelyusedduetoitssimplicityand nondestructivenature.Itworksbytakingintoaccounttheovertoneand combinationabsorptionbands.Theabsorptionhereisweakandoccursas aseriesofoverlappingbonds.Becausetheabsorptionbandsareweak,they arehighlyusefulinanalyzingsamplesthatarestrongabsorbersandhavehigh lightscattering(BellandXu,2018; El-Azazy,2019; Ewingand Kazarian,2017). 3 Introductiontochemicalanalysisofforensicsamples
2.1.3Nuclearmagneticresonancespectroscopy
Nuclearmagneticresonance(NMR)spectroscopyisaninterestinganalyticaltechniquethattakesintoaccountthemagneticmomentofnucleispinin thepresenceofanexternalmagneticfieldforthestructuraldeterminationof achemicalcompound.Typically,nucleipossessaneutronandprotonsthat contributetothespinofthenuclei.Ifanexternalmagneticfieldisapplied thenbasedontheenergylevelofthenucleispin,thereisanenergytransfer fromthegroundenergyleveltothehigherenergylevel.Thisoccursata particularwavelengthandfrequencyandischaracteristictothecompound. Typically,HNMRisusedasthehydrogennucleusiscomposedofonlya singleproton,thusresultinginbetterspectra.However,apartfrom HNMR,phosphorousNMRisalsousedfortheanalysisofdifferentforensicevidence.NMRinforensicscienceistypicallyusedtoanalyzeillicit drugs,explosives,poisonsandtoxins,andbodyfluidssuchasblood,semen, andsaliva.Ithasalsobeenusedfortheanalysisofpostmortemchanges. NMRisahighlysensitivetoolthatiscapableofnotonlyelucidatingthe structuraldetailsofacompound,butcanalsoeasilyidentifyanyimpurities, thusdeterminingquality.ThesefeaturesmakeNMRahighlycriticaltool fortheanalysisofforensicevidence(ElipeandVictoria,2012a,b).
2.1.4Ramanspectroscopy
Ramanspectroscopyisyetanothervibrationalspectroscopytechnique,and itismainlyusedforthestructuralanalysisofacompound.Inthistechnique, Ramanscatteringservesasthemainbasisofanalysis.TypicallyinIRspectroscopy,thesampleisirradiatedwithinfraredradiation.Whentheenergy ofthemoleculematchesthatoftheradiationsource,absorptionoccurs,followedbythevibrationofthemolecularbonds.However,inRamanscattering,theradiationsourceistypicallyalaserthathasasinglewavelengthand frequency.Oncetheradiationhitsthesample,theelectroncloudaroundthe nucleiinelasticallyscatterstheradiation,duetowhichtheenergyofthescatteredphotonisdifferentfromthatoftheincidentphoton.Thisdifference correspondstoonevibrationalunit;thistypeofscatteringisknownas Ramanscattering.Ramanscatteringhastwotypesofshifts:thestokes andtheantistokesshift.Thestokesshiftoccurswhentheelectrontransfers fromalowerenergyleveltoahigherenergylevelwhiletheantistokesshift occurswhentheelectronmovesfromahigherenergyleveltoalower energylevel.Duetothesephenomena,thereisadifferenceinthefrequency andwavelengthofthemoleculethatischaracteristicofitsstructure.In forensicscience,Ramanspectroscopyisusedforitseaseofuseand
nondestructivenature.Samplessuchasblood,saliva,semen,fingerprints, illicitdrugs,paints,pigments,explosives,andgunshotresiduearewidely used.Itisalsousedfortheon-spotdetectionofcounterfeitdocuments (Fikietetal.,2018; JohnandGeorge,2017).
2.1.5Atomicemissionandabsorptionspectroscopy
Atomicabsorptionandemissionspectroscopyareatomicspectratechniques thatconsiderabsorptionandemissionbyatomswhentheyaresubjectedtoa monochromaticlightsource.Thesetechniquesareexcellenttoperformthe elementalmappingofasample.Inatomicabsorptionspectroscopy,amonochromaticradiationsourceemittedbytheatomizationoffreeatomsis absorbedbythesample.Becauseatomicabsorptionspectroscopyisbased ontheabsorptionoftheradiationemittedbythesource,itfollowstheBeer Lambertlaw,whichcorrelatestheamountofradiationabsorbedwiththe concentrationoftheelementinthesample.Throughthis,itispossibleto obtainthecharacteristicintensitiesoftransmittedradiation.Therefore,it canbeusedforthequantitativedeterminationoftheelementinthesample. Inatomicemissionspectroscopy,whentheradiationhitsthesample,the electronsabsorbsomeoftheradiationandtransitiontoahigherenergylevel. Onceexcited,theytendtocomebacktotheirgroundstate,whichthen leadstotheemissionofradiationofawavelength.Thisemissionischaracteristicoftheelementpresentinthesample.Quantitativeanalysiscanalsobe performedbecausetheamountofemittedradiationisproportionaltoits concentration.Becausethesetechniquesareprimarilyusedforthequantitativeestimationandmappingofelementsinthesample,itisusedinforensic sciencemainlyfortheanalysisofexplosives,gunshotresidue,andother ammunition.Evensamplessuchasfibersandflammablematerialscanalso beanalyzedthroughthistechnique(Cantle,1982; Moore,1989).
2.2Chromatographictechniques
Chromatographywasfirstdevelopedin1900byaRussianscientist,Mikhail Tsvett.Itwasinitiallyusedtoseparatetheplantpigmentsinaleaf.Itisbasicallyaseparationtechniquethatemploysastationaryphaseandamobile phase.Theseparationtakesplacesonthebasisoftheaffinityoftheanalytes towardeitherthestationaryphaseorthemobilephase.Typically,theanalytesgetadsorbedonthestationaryphaseiftheyhavemoreaffinitytoward it,duetowhichtheytravelslower.Iftheyhavehigheraffinitytowardthe mobilephase,theytendtotravelfaster.Basedonthisbasicprinciple,the analytesinanycompoundcanbeeasilyseparatedandidentified.Substances
thattravelslowlyhaveahigherretentiontimewhilethosethattravelfaster havealowerretentiontime.Inthissection,wediscussthevarioustypesof chromatographictechniquessuchashigh-performanceliquidchromatography(HPLC),gaschromatography,andhigh-performancethinlayerchromatography(HPTLC)aswellasofferabriefoverviewofhyphenated techniques.
2.2.1Highperformanceliquidchromatography
HPLCisatypeofcolumnchromatographytechniqueinwhichthestationaryphaseisinacolumnandthemobilephaseispassedthroughitunderhigh pressure.TheentireprocessinHPLCisautomatedandcurrentlyseveral instrumentscomewithautosamplersaswell.Thestationaryphaseisofmany typesinHPLC,includingnormalphase,reversephase,ionexchange,and sizeexclusion.InnormalphaseHPLCcolumns,thestationaryphase,which isusuallysilicagel,ismorepolarthanthemobilephase.Inreversephase columns,thestationaryphaseisnonpolar,andthemobilephaseusedispolar. Ionexchangecolumnsandsizeexclusioncolumns,useacidic/basiccolumns orporousstationaryphases,respectively.Illicitdrugssuchasopioidsand cannabisaswellaspesticides,poisons,planttoxins,andalkaloidsareanalyzed usingHPLC.Theanalysisofeachsampledemandsitsownprotocolwherein factorssuchasthetypeofcolumntobeused,themobilephase,thepHofthe mobilephase,flowrate,retentiontime,andpressureareconsidered.The detectorsusedinHPLCalsovaryaccordingtothetypeofsampletobeanalyzed.CommonlyuseddetectorsareUV/visible,fluorescence,refractive index,electrochemical,conductivitydetector,evaporativelightscattering detector,andchiraldetectors(Ahuja,2005; Daldrupetal.,1986; Ghosh,1992).
2.2.2Gaschromatography
Gaschromatographyisyetanotherinterestingchromatographytechnique thatiswidelyusedinforensiclaboratories.Inthistechnique,themobile phaseisanobleorunreactivegassuchasargonorhelium.Itispassed throughthestationaryphase,whichispresenteitherasapackedcolumn orcoatedinsideacapillary.Inthistechnique,thesampleiscarriedalong withthecarriergasorthemobilephaseandispassedthroughthecolumn. There,basedontheaffinityofthesampletowardthestationaryorthe mobilephase,itwillbeseparatedatdifferentretentiontimes.Whilethis isahighlyversatiletechnique,ithascertaindrawbacks.Oneisthatthistechniqueisunsuitableforthermallylabilecompounds,asitcangetpyrolyzed
whenthesampleisconvertedtothevaporstatepriortoanalysis.However, withpropersamplepreparationtechniquesandtheuseofprotectantsto ensuresampleintegrity,itisnowpossibletoaccuratelyseparatethecompoundsinasample.Illicitdrugsandbloodalcoholcontentaresomeof thewidelyanalyzedforensicsamples.Apartfromthis,explosivesandother flammablesubstances,accelerants,andevenpesticidesandpoisonscanalso bedetectedthroughthistechnique(Blumberg,2012; deConingand Swinley,2019).
2.2.3Highperformancethinlayerchromatography
High-performancethin-layerchromatographyisprobablyoneofthemost versatilechromatographictechniquesusedfortheanalysisofforensicevidence.Inthistechnique,athin-layerchromatographyorTLCplatecoated withtheadsorbentorthestationaryphaseisusedandincubatedinachamber saturatedwiththemobilephase.Thesampleisalsocarefullyloadedonthe TLCplatepriortothechromatographicdevelopment.Theretentiontime ofthesamplesinthiscaseiscalculatedaccordingtothedistancetraveledby theanalytespresentinthesample.Thistechniqueishighlyusefulinforensic scienceasitoffersphotographicproofoftheevidencethatcanbeeasilypresentedincourt.Also,everystageinthistechniqueisindependentofeach other,duetowhichanycumulativeerrorsareminimized.Whilesamples suchasillicitdrugsandtoxinsarecommonlyanalyzedusingthistechnique, samplessuchasfoodadulterants,ink,fabriccounterfeiting,andevenexplosivescanalsobedetectedusingthistechnique.Thistechniquealsooffers excellentautomation,optimization,anddetectionwithminimumsample preparation(Bensakhria,2017; Srivastava,2011).
2.2.4Hyphenatedtechniques
Whilechromatographictechniquesareusefulfortheseparationofanalytes andtheirdetectionisfoundthroughtheirretentiontime,itissometimesnot possibletoaccuratelydeterminethestructureofthecompound.Therefore, thesetechniquesareoftenpairedwithothertechniquesthatarecapableof elucidatingthemolecularstructure.GC,HPLC,andHPTLCareoften pairedorcoupledwithtechniquessuchasamassspectrometer(MS),FTIR, andNMR.GC-MSisatechniqueinwhichthesampleisseparatedasusual intheGC.Oncetheyareseparated,aspecificdeviceisusedtocouple thetwoinstruments,throughwhichthesamplesfromGCaretransported totheMS.TheMSthenelucidatesthestructureofthemolecule.Similarly, inthecaseofLC-MS,onceseparatedbythechromatographictechnique,
thecompoundsundergostructuralanalysisbytheMS.Similarly,HPTLC canalsobepairedwithFTIRandMS.Acrucialaspectofsuchhyphenated techniquesisthelinkthatconnectsthetwo.Becausebothtechniquesuse differentsampleforms,itisimportantthatappropriatesamplepreparation techniquesareused(Bogusz,1999; Cimpoiu,2011; Kitsonetal.,1996).
2.3Microscopictechniques
Microscopictechniquesareamongthemostwidelyusedtechniquesforthe preliminaryandconfirmatoryanalysisofforensicsamples.Whilespectroscopicandchromatographictechniquesarecapableofobtainingaccurate structuralandchemicalinformation,theydonotprovideanactualvisualizationofthecompound.Microscopictechniquesovercomethislimitation andprovidehardresultsthatcanbeeasilyproducedincourt.Inthissection, thedifferentelectron,optical,andprobemicroscopesarediscussed.
2.3.1Opticalmicroscopes
Opticalmicroscopesareprobablyamongthemostversatileandubiquitous toolsusedforthevisualizationofforensicevidence.Opticalmicroscopes visualizesamplesbyhittingthesampleswithalightsource,whichgenerates photons.Thesephotonsinteractwiththesampleandthereflectedandscatteredlightarecollectedbyaseriesoflenses.Therearemainlytwotypesof opticalmicroscopes,thesimpleandthecompoundmicroscope.Thesimple microscopeconsistsofasinglelenswhilethecompoundmicroscopeconsists ofaseriesofobjectiveandcondenserlenses.Mostmicroscopesusedina forensiclaboratoryarecompoundmicroscopes.Acompoundmicroscope consistsofalightsource,acondenserlensthatisresponsibleforcollecting andfocusingthelightobtainedfromthelightsourceontothesample,andan objectivelensthatfocusesandcollectsthelightfromthesample.Typically, phasecontrast,brightfield,fluorescence,polarized,andstereomicroscopes areamongthemostcommonlyusedmicroscopesfortheanalysisofvarious evidence.Whileanybiologicalsamples,explosives,andammunitionare commonlyanalyzedusingthemicroscopes,samplessuchasfibers,glass, paints,andevenquestioneddocumentscanbeeasilyanalyzedthroughopticalmicroscopes(Haynes,1984; Johnson,2013).
2.3.2Electronmicroscopes
Electronmicroscopessuchasscanningelectronmicroscopesandtransmissionelectronmicroscopesareamongthemostadvancedmicroscopescapableofvisualizingacompoundwithextremelyhighresolutionand
magnification.SEMisusedtoanalyzethesurfacemorphologyofamaterial whileTEMisusedtovisualizeitsinternalmorphology.SEMiscapableof visualizingthesurfacemorphologybyanalyzingthebackscatteredandthe secondaryelectrons,whichareproducedwhenthebeamoftheprimary electronshitsthesame.Thebackscatteredandsecondaryelectronsaregeneratedasaresultoftheimpingementoftheincidentelectronsonthesample. InTEM,theprimaryelectronbeamismadetopassthroughanultrathin sampleandonlythetransmittedelectronsaredetected.Basedonthe obstructionspresentinsidethematerial,someoftheelectronsgetscattered, duetowhichthoseareasappeardarkerintheimage.Duetosuchamodeof functioning,SEMrequiressamplesthatareconductiveinnaturesothatthe primaryincidentelectronscaneasilyinteractwiththesurface.Therefore, priortousage,thesamplesarecoatedwithametalsuchasgold.InTEM, thesamplesmustbeultrathininordertoensurethattheelectronscanpass througheasily.Inforensicscience,SEMhasfoundmoreusagethanTEM duetothenatureoftheevidenceobtained.Biologicalevidencesuchashair, pollen,teeth,skin,orbonecellscanbeeasilyimagedthroughthesetechniques.Themicroscopicanalysisofsuchevidenceitselfisconfirmatoryin nature.Apartfromthis,variousanthropogenicandgeologicalsampleshave alsobeenanalyzedusingthistechnique(Amelinckxetal.,1997; Fultzand Howe,2013; Goldsteinetal.,2003).
2.3.3Atomicforcemicroscope
Atomicforcemicroscopy(AFM)isatypeofscanningprobemicroscope usedtoproducethree-dimensional(3D)imagesofthesamplesurface.Inthis technique,aprobeorsmalltipmovescarefullyalongthesurfaceofthesampleinordertogeta3Dmorphologyofthesurface.Theworkingofthis microscopeissimilartothatofablindmanwalkingontheroadwithhis stick.Theprobeisusuallyattachedtoacantileverthatcapturesthemovementsoftheprobe.Alaserlightfallsonthesurfaceofthetipinorderto monitoritsmovement.Therearebasicallythreetypesofworkingmodes inAFM:contactmode,noncontactmode,andtappingmode.Incontact mode,thetiportheprobeisinconstantcontactwiththesamplesurface andbasedontheforcesdetectedbythetiponthesamplesurface,the3D morphologyisobtained.Innoncontactmode,thetipisnotincontactwith thesurfaceofthesampleandagainmovesbyconsideringaminimumthresholdofforcedeflectiononthesamplesurface.Intappingmode,rasterscanningoccurswhilethetipisconstantlyoscillatingtoandfrofromthesample surface.Dependingonthetypeofsampletobeanalyzed,thedifferent
workingmodescanbeselected.Inforensicscience,however,theuseofthis microscopeisstillnotwidespread,anditremainsintheresearchstages. However,thisisahighlypromisingtechniqueasitoffersa3Dsurfaceanalysisofthesample(Pandeyetal.,2017b).
2.3.4EnergydispersiveX-raycoupledmicroscopyinforensicscience
EnergydispersiveX-ray(EDX)isatechniquecommonlyusedfortheelementalanalysisofamaterial.Inthistechnique,thesampleisirradiatedwith anelectronbeam,duetowhichbackscattered,auger,andothersecondary electronsaregenerated.Theseelectronsaretypicaloftheelementspresentin thesample,duetowhichquantitativeandqualitativeelementalinformation canbeeasilyobtained.TheEDXisusuallycoupledwithelectronmicroscopeswheretheelectrongunofthemicroscopeitselfactsasthesource forEDXaswell.TheEDXdetectorisusuallykeptclosetothesamplein ordertoefficientlycapturethescatteredelectrons.Typically,anEDXdetectorconsistsofanSi-lithiumdetectororasilicondriftdetector.Justlikeinan electronmicroscope,theacceleratingvoltageoftheelectronbeamandthe thicknessofthesampleplaycrucialrolesindeterminingtheelementalinformation.Inforensicscience,EDXhasbeenmajorlypairedwithSEMtoanalyzesamples.Typicalforensicevidencesuchasbiologicalsamples, explosives,andtoxinsareroutinelyanalyzedthroughthistechnique.Apart fromthis,paints,glass,andgunshotresiduearealsoanalyzedthroughthis technique(AbdMutalibetal.,2017; Hodoroaba,2020).
2.4Emerginganalyticaltechniques
Spectroscopic,microscopic,andchromatographictechniquesareroutinely usedinforensiclaboratoriesacrosstheworld.Currently,thesetechniques haveseenanincreaseintheirsupplementationwithemerginganalytical techniquessuchasmassspectrometry,X-raydiffraction,lab-on-chip devices,andnanotechnology.Inthissection,abriefoverviewofsuchtechniqueswillbediscussedalongwithhowtheyhavebeenusedfortheanalysis ofvariousforensicevidence.
2.4.1Massspectrometry
Massspectrometry,asmentionedbefore,iscapableofanalyzingorganic compoundsbyvaporizingthesamplesintoagasphase,thenseparating theionsaccordingtotheirmass/chargeratio(m/z).Typically,amassspectrometerconsistsofanionsourcethatisresponsiblefortheionizationofthe samples,amassanalyzerwheretheionsareseparatedaccordingtothem/z
Introductiontochemicalanalysisofforensicsamples
ratios,andadetectorthatdetectstheseparatedions.Thistechniqueiscapableofnotonlyobtainingstructuralinformationbutalsoperformingaquantitativeestimation.Bothliquidandsolidsamplescanbeeasilyanalyzed throughthistechnique.Atypicalmassspectrumconsistsofthem/zratio valuesonthe x-axisandtheirrelativeintensitiesonthe y-axis.Thestrongest peakcorrespondstotheparentionofthesample,whichisbasicallytheionizedformofthesamplewiththemolecularweightclosesttoituponfragmentation.Therearemanyionizationmethodssuchaselectronionization, chemicalionization,fieldormatrixdesorption,atmosphericpressurechemicalionization,plasmadesorption,thermosprayionization,andfastatom bombardment.Massanalyzerssuchasaquadrupoleanalyzer,timeofflight (TOF),andiontraparecommonlyused.Detectorssuchasphotomultiplier tubes,Faradaycups,andscintillatorsarealsoused.Typically,massspectrometersareoftencoupledwithliquid,gas,orthinlayerchromatographictechniquesinordertogetanaccurateanalysisoftheforensicevidence.Heretoo, thistechniqueisusedfortheanalysisofmyriadforensicevidencesuchas biologicalsamples,illicitdrugs,andexplosives.Gunshotresidueandcounterfeitinkscanalsobeanalyzedthroughthistechnique.Thisisahighlyversatiletechniqueandthereforebothsolidandliquidsamplescanbeeasily analyzed(deHoffmannandStroobant,2007).
2.4.2X-raydiffraction
X-raydiffraction(XRD)isatechniqueusedtoanalyzethecrystallinityofa material.InX-raydiffraction,whenX-rays,preferablyofhigherenergy,hit theatomsthatarearrangedinamaterial,theyundergotwotypesofscattering:elasticorinelastic.Inelasticscattering,thescatteredrayshavethesame energyastheincidentelectronwhileininelasticscattering,theenergyofthe scatteredraysisnotequaltotheenergyoftheincidentrays.Duetothis, thereisaconstructiveordestructiveinterferencebasedonthecrystalorientationofthesample.TheconstructiveinterferencefollowstheBragglaw, whichtakesintoaccounttheanglebetweentheradiationandthecrystal planes,thewavelengthoftheradiation,andthespacingbetweenthecrystal planes.ThroughtheBraggequation,itispossibletoobtaininformation regardingthecrystallinityofthesample.Typically,theanalysistakesplace eitherbyfixingorvaryingtheanglebetweentheincidentrayandthecrystallineplanes.Metalssuchascopper,molybdenum,orironarecommonly usedtogeneratetheX-raysourcesbysubjectingthemtoahigh-voltage electronbeamundervacuumconditions.Thesamplescaneitherbyanalyzedinapowderedformorasawholecrystal.Basedonthetypeofsample
tobeanalyzed,thetypeofX-raydiffractionmethodcanalsobeselected. Scintillation,solidstate,orchargecoupleddevicesareoftenusedasdetectors inthistechnique.XRDhasbeencommonlyusedtoanalyzethecrystalline structureofdrugs,minerals,andothermetals.Butinforensicscience,ithas alsobeenusedtoanalyzepaints,pigments,bones,andevenfibers(Bishnoi etal.,2017; Br € ugemannandGerndt,2004).
2.4.3Nanotechnology
Nanotechnologyisthescienceofnanomaterials,thatis,thosematerials whosesizeisinthenanometerrange.Duetotheirsmallsize,theyhave enhancedoptical,magnetic,andelectricalpropertiesalongwithincreased reactivitycontributedbyasignificantincreaseintheirsurfacearea.Due tosuchproperties,nanomaterialshavefoundmyriaduseinvariousapplications.Nanomaterialssuchasnanoparticles,nanotubes,nanoshells,andnanofibersareoftenusedinvariousapplications.However,suchnanomaterials areoftenfunctionalized,thatis,thesurfacesofthesenanomaterialsareoften modifiedbyincorporatingaguestmoleculeorafunctionalgroup.Dueto functionalization,theintrinsicpropertiesofthenanomaterialsgetfurther enhanced.Nanomaterialsarecommonlysynthesizedthroughtworoutes: thebottom-uporthetop-downapproach.Inthebottom-upapproach,a precursorsaltsolutionistakenandinthepresenceofacatalystorareducing agent,nanomaterialsareformed.Inthetop-downapproach,thebulkmaterialisgroundtonanomaterialsthroughvariousmechanicalandchemical methods.Thecharacterizationofthesenanomaterialsisdonethroughvariousspectroscopic,electron,andprobemicroscopictechniques.TheelectronmicroscopesareusuallypairedwithEDXtogetelementalinformation. Inforensicscience,nanomaterialshavebeenwidelyusedforthedevelopmentofsensorsthatallowthedetectionofexplosives,gunshotresidue, andothertoxinsandpesticides.Duetotheirnanosize,theyhavealsobeen usedforthedetectionoflatentfingerprintsastheycaneasilygetattachedto theridgesofafingerprint,thusallowingenhancedselectiveandsensitive detectionofthefingerprintswithincreasedcontrast(Pandeyetal.,2017a; Rawtanietal.,2019; Tharmavarametal.,2018).
2.4.4Lab-on-chipdevices
Lab-on-chipdevicesareamongthemostadvanceddevicescreatedforthe analysisofforensicsamples.Thesedevicesareminiaturizedlaboratoriesand aredevelopedthoughmicrofluidictechnology.Thesearebasicallyanamalgamationoffluidics,electronics,andoptics.Thesedevicescancarryout
independentreactionsthatusuallyneedanentirelaboratorytowork.Dueto this,theamountofsamplerequiredisverysmall(microliterstonanoliters) andthereactionisveryrapid,duetowhichon-spotdetectionkitscanbe easilydeveloped.Alab-on-chipdevicetypicallyconsistsofaninjectoror asampleholder,anditisconnectedtoasampleprocessingchamberwhere anycontaminantsareremovedthroughcentrifugationorfiltration.Thisis followedbyareagentmixerthatservesasthemaincomponentformixing thereagents.Oncethisisdone,itissenttothereactionchamberwherethe sensorsareattachedtomonitortheproductformationtoanalyzethereactions.Thedevicealsoconsistsofatransducerthatisresponsibleforconvertingthechemicalsignalstoelectricalsignalsandconvertingthatintoa readableoutput.Lab-on-chipdevicesarehighlyusefulforanalyzingforensic evidence,astheyrequireverysmallamountsofsamplesandallowthe on-spotdetectionofevidencewithhighspecificity.Whiletheuseoflabon-chipdevicesfortheanalysisofevidenceisstillmostlyintheresearch stage,itholdsimmensepotentialforuseinthefield.Theycanbeused fortheanalysisofDNA,illicitdrugs,andevenexplosives.Theycanalso beusedfortheanalysisofvariousbiologicalevidencesuchasbloodand caneasilydetectthedifferencebetweenanyred-coloredliquidandactual blood.Theuseoflab-on-chipdevicesinthefieldwillsignificantlyreduce thetimerequiredforacriminalinvestigation,duetowhichvariousproactiveforensicmeasurescanalsobeimplemented(AbgrallandGue,2007; Guptaetal.,2016).
3Conclusion
Theanalysisofvariousforensicevidenceishighlynecessary,asthiscanform thecruxofanycriminalinvestigation.Apartfromcriminalinvestigation,the analysisofvarioussamplesisalsorequiredtoimplementsecuritymeasures andlaws,especiallyinairports.Overtheyears,samplessuchasillicitdrugs; explosives;gunshotresidue;biologicalevidencesuchasblood,semen,saliva, DNA,pollen,anddiatoms;andotherphysicalevidencesuchaspaints,pigments,fabrics,andquestionabledocumentshavebeenanalyzedusingvariousanalyticaltechniques.Inthischapter,thevariouschemicalanalytical techniquesfortheanalysisofvariousforensicsampleshavebeendiscussed inbrief.SpectroscopictechniquessuchasUV-visible,infrared,Raman, nuclearmagneticresonanceandatomicemissionandabsorptionspectroscopieshavebeenusedforthepreliminaryandconfirmatoryanalysisofevidencealongwithchromatographictechniquessuchashigh-performance
liquidchromatography,gaschromatography,andthin-layerchromatography.Thesetechniquesareusuallypairedwithamassspectrometertoobtain informationregardingstructure.Typically,theanalysisofevidenceisintandemwithopticalandelectronmicroscopictechniquesthatprovideanaccuratevisualizationofthesample.Apartfromsuchconventionaltechniques, emerginganalyticaltechniquessuchastheuseofnanotechnologyand lab-on-chipdevicesalsohaveimmensepotentialintheanalysisofvarious forensicevidence.
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Forensicsamplingandsample preparationtechniques
1Introduction
Aforensicsampleisevidencecollectedfromthesceneofacrime.Thisevidenceprovidesinformationaboutthecrimeinmanywayssuchasindividual testimony,documentedproof,orthroughthesubmissionofmaterialobjects incourtroomtrials.Onabroaderscale,evidencecanbeoftwotypes:real anddemonstrative.Realevidenceisdirectlyrelatedtoandcollectedfrom thecrimescene,anditcanassistinidentifyingthevictimorthesuspect. Mostforensicsamplescollectedfromthecrimescenesuchasbiological fluids,explosives,gunshotresidue(GSR),drugs,toxins,alcohol,etc.,are realevidence.Ontheotherhand,demonstrativeevidenceisnotdirectly collectedfromthecrimescene.Thisisdevelopedatlaterstagesofforensic investigationinordertoassistandunderstandtheimportanceofrealevidence.Examplesofsuchevidenceincludeadigitallysimulatedcrimescene, figuresdemonstratinghaircharacteristics,bloodstainpatterndemonstration, etc.Evidencecollectedfromthecrimescenecanbecircumstantial,conclusive,conflicting,corroborating,derivative,exculpatory,foundational,hearsay,incriminating,presumptive,probative,rebuttal,ortainted(Houckand Siegel,2010).Asfarasthetransferofevidenceisconcerned,itcanbedirect (frompersontopersonortoalocation)orindirect(involvementofintermediatepersonsorobjects).Inindirecttransfer,thechancesofcontaminationofforensicsamplesareveryhigh.Atacrimescene,normallythree modesofsearchesareperformedfortheidentificationandcollectionof forensicsamples.Thesesearchpatternscanbelaneorstrip,spiral,andgrid (HouckandSiegel,2010).Usingthesesearchpatterns,differentkindsof forensicsamplesareproperlycollected,labeled,andforwardedinasuitable mannertothelaboratoryforfurthertesting.
Inthischapter,differenttypesofforensicsamplesbelongingtothree differentcategories—physical,chemical,andbiological—areinitiallydiscussed.Thechapteralsodiscussesthecollection,preservation,andhandling
ofsuchforensicsamples.Further,thepreparationtechniquesinvolvedfor theexaminationofforensicsamplesthroughvariousanalyticalinstruments arealsoelaborated.
2Typesofforensicsamples
Broadly,theforensicsamplescollectedfromacrimescenecanbeclassified intothreecategories:physical,chemical,andbiological(Robertson,2016). Thissectiondiscussesthetypesofforensicsamplesfallingintothesecategoriesaswellastheircollection,preservation,andhandling.
2.1Physicalforensicsamples
2.1.1Classification
Physicalforensicsamplescanbeclassifiedmainlyintofivecategories:questioneddocuments,digitalsamples,impressionmarks,ballisticssamples,and tracesamples.Thelatterthreecategoriesincludealargenumberofforensic samples.Impressionmarkscollectedatthecrimesceneincludelipprints, fingerprints,footprints,andtireandtoolmarks.Forensicsamplesrelated toballisticsincludefirearms,GSR,bullets,andcartridgecases.Traceforensicsamplesmainlyincludesoil,paint,fiber,andglass.Thesearethedifferent typesofphysicalforensicsamplesthatareencounteredduringcrimescene investigations(Collett,2005).
2.1.2Collection,preservation,andhandling
Thecollection,preservation,andhandlingofdifferentphysicalevidence vary.Asfarasdocumentsareconcerned,theycanbefoundeitherintact orinatornorcharredstate.Torndocumentsshouldbecollectedbyswitchingoffthefanandpickingthemup,preferablywithforceps.Theyshouldbe properlyarrangedonaglassorplasticsheetduringreconstruction.Inthe caseofcharreddocuments,iftheyarepresentinacontainer,thentheentire containershouldbecollected.Iftheyarepresentinaheapwithunburned papersinside,thentheentireheapshouldbecollectedandsenttothelaboratory.Allthecollecteddocumentswhethercharred,torn,orintactmust bepreservedafterphotographyinacardboardorplasticboxduringtransportationtothelaboratory.
Inthecaseofdigitalevidence,ifevidenceispresentinsmalldevicessuch asacellphoneortablet,thenitcanbedirectlycollectedusinggloves.If, however,itispresentindevicesthatcannotbemovedsuchasadesktop computer,thentheevidencemustbecollectedandpreservedona
19 Forensicsamplingandsamplepreparationtechniques
CD-RorDVD.Inthecaseofswitched-offsystemssuchasalaptopordesktopcomputer,theharddrivemustbecollectedandsignedforbytheuser andofficerincharge.Also,nearbyplacesmustbesearchedforanypaperor diarythatmayhavethepassword.Inthecaseofswitched-onsystems,data mustbecollectedthroughdigitalforensictools.Ifnoexpertisavailable,then thepowersupplyshouldberemovedfromthesystemandtheharddisk shouldberecovered.Allthedigitalsamplesmustbephotographedandsent tothelaboratoryinantistaticpackingmaterialsuchasapaperbag,cardboard box,orenvelope(Casey,2004).
Impressionmarksincludefingerprints,lipprints,footprints/shoeprints, andtireandtoolmarks.Latentfingerprintsafterdevelopmentwithfingerprint powderareliftedbyadhesivetapeandpreservedonglassslidesfortransportation.Inthecaseofpatentorplasticfingerprints,theobjectisphotographed andtakendirectlyforlaboratoryanalysis.Lipprintsarealsocollected,preserved,andtransportedinasimilarfashion.Photography,lifting,tracing, andcastingarethemethodsforthecollectionoffootprints/shoeprintsortire marks.Castingcanbedonebyresin,plasterofParis,wax,etc.Adhesivetape andgelatinliftersareusedtoliftimpressionmarksfromdifferentsurfaces.Tool marksarecollectedeitherbycollectingtheentireobject,orbycastingusing plasticorrubber(softened)onthesurface.Thephotographsinthiscasemust betakenperpendiculartothetoolmarks(Robertson,2016).
Firearms,cartridges,andGSRaretheballisticforensicsamplesfoundat thecrimescene.Ifafirearmisrecovered,thenitshouldbesafelycollected afterunloadingandarecordoftheliveandfiredcartridgesshouldbekept.It shouldbepackedinacellophanepacketifanyforeignmaterialsuchas blood,hair,ortissueispresentonthefirearm.Thefirearmshouldbetransportedinawoodenboxwithsufficientpadding.Inthecaseofammunition, partssuchaslivecartridges,dischargedbullets,bulletfromthecadaver,etc., shouldbesafelyrecoveredandpackedseparately.GSRiscollectedusinga castofmoltenwaxonthesuspect’shand.IfGSRisonclothes,thenthe entirematerialshouldbepackedincellophanepacketsandsenttothelaboratoryforfurthertesting(Saferstein,2013).
Glass,fibers,paints,andsoilsarethetraceforensicsamplesfound.All glassparticlespresentatthecrimescenemustbecollectedseparatelyand keptincottonpadding.Ifglassispresentinanobjectwhichcanbetransportedsuchasashoeorawindow,thentheentireobjectshouldbetransportedforanalysis.Inthecaseoffibersorthreads,theyshouldbecollected usingtweezersandplacedinenvelopesorpaper,thensealedinanother envelope.Duringpaintcollectioninhit-and-runcases,thesampleshould
becollectedfromthefreshlydentedregionsofthevehicle.Inthecaseof burglary,theareawherethetoolwashitshouldbeusedforpaintcollection andthetoolshouldalsobesealedwithpaperandtapefortransportation.Soil samplescanbecollectedbyvacuumingandpackinginplasticcontainersfor transporttothelaboratory(Saferstein,2013).
2.2Chemicalforensicsamples
2.2.1Classification
Chemicalforensicsamplescanbeclassifiedasdrugs,toxins,fireandarson samples,explosives,andpetroleumproducts.Drugsincludesamplessuch asnormalonesusedathighdoses,injections,andnarcoticandpsychotropic drugs.Toxinsaremainlypesticides,alcohols,orpoisons.Explosivesamples canbeintact,residue,orasampleofsoilwithanexplosive.Inthecaseof petroleumproducts,furtherclassificationincludesproductssuchaskerosene,diesel,andbiofuel.Thesedifferentkindsofchemicalevidencearecollectedandpreservedindifferentmanners(HouckandSiegel,2010).
2.2.2Collection,preservation,andhandling
Drugs(solidsandpowders)aregenerallycollectedininertdrycontainersand transportedincustomizedtabletboxes.Preservativesarenotadded;however,refrigerationismaintainedifrequired.Plasticvialsorpillboxesareused forcollectingcounterfeittablets.Abuseddrugsandpsychoactivesubstances areseizedinheat-sealedbagsofplasticforsolidsandplasticorglasscontainersforliquidsamples,whichshouldbewrappedincleancloth.All thecontainersneedtobeproperlylabeledbeforetransporttothelaboratory (HouckandSiegel,2010).
Inthecaseofalcoholpoisoning,bodyfluidssuchasbloodandurineneed tobecollected.Bloodshouldbecollectedfromthesubclavianorfemoral vesselinananticoagulantcoatedvial,andtransportediniceboxes.Inthe caseofurine,thereisnorequirementofapreservative.Incasesoffatalpoisoning,visceralorgansarecollectedwithdifferentpreservatives.Arectified spiritinacidpoisoningandacommonsaltorsaturatedsolutionofitincases ofalcohol,carbonmonoxide,cyanide,orsnakevenompoisoningareusedas preservativesforthetransportationofviscera.Commonsaltwithpotassium oxalateisusedforpreservingbloodinpoisoningcases.
Incasesoffireandarson,samplessuchascigarettes,matchsticks,andcontainersofflammableliquidsareofforensicimportance.Theseshouldbe carefullycollected.Flammableliquidscanbecollectedusingaportable vapordetector.Soilsampleswithtracesofflammableliquidsshouldalso
